Project Summary Altered glucose metabolism is a common cancer phenotype. Because cancer cells and normal cells use metabolism differently, targeting this difference holds promise for improved cancer therapy. However, our understanding of how cancer cells incorporate nutrients into biomass to support growth and proliferation is based primarily on studies of cultured cells. Nutrient levels in culture differ substantially from those experienced by cells in tumors and these differences have a dramatic effect on metabolic phenotypes. Accordingly, we are using relevant mouse models of human cancer to examine the biochemical underpinnings of how glucose metabolism contributes to the initiation, growth, and progression of cancer. A key step in glucose metabolism is catalyzed by the glycolytic enzyme pyruvate kinase. Most mammalian cells express either the PKM1 or the alternatively spliced PKM2 isoform of this enzyme. PKM1 has high constitutive activity and favors the oxidation of glucose-derived pyruvate. Cancer cells universally express PKM2, which can adopt either an active or inactive state. Regulation of pyruvate kinase determines whether cells can synthesize enough nucleotides to proliferate, with low pyruvate kinase activity favoring a metabolic state that allows proliferation. This leads to several unanswered questions, including whether any pyruvate kinase is needed for tumor cell proliferation, how pyruvate kinase activity affects nucleotide synthesis, and how to practically use this information to improve cancer treatment. We are taking advantage of the fact that PKM1 expression and high pyruvate kinase activity is tumor suppressive to study how glucose metabolism affects nucleotide synthesis. By manipulating pyruvate kinase isoform expression or the availability of key metabolite precursors, we aim to determine the requirement for pyruvate kinase in cancer, and dissect how metabolism is altered by pyruvate kinase activity to affect nucleotide synthesis. For these studies we will take advantage of unique mouse models and small molecule drugs to manipulate tumor metabolism. In Specific Aim 1, we will answer the question of whether pyruvate kinase is dispensable for proliferation in a tumor. In Specific Aim 2, we will test the hypothesis that pyruvate kinase affects the redox state of cells, and that this is the mechanism by which pyruvate kinase isoform expression affects tumor metabolism and growth. In Specific Aim 3 will perform preclinical studies in a prostate cancer model to determine how to use pyruvate kinase activators to treat patients. Together, these studies will advance our understanding of glucose metabolism and how it supports nucleotide synthesis and cancer progression. This work will also inform the design of future clinical trials and identify approaches to target metabolism for cancer therapy.